Determining the structure and imaging the dynamical behavior of large protein complexes as well as other biological nanoparticles at room temperature with near atomic resolution has the potential to greatly impact structural biology and our knowledge of biomolecular function and interactions. A major bottleneck in structural biology is that many of the proteins performing critical cellular functions are membrane proteins that have proven intractable to structure determination by traditional x-ray crystallography, in which x-ray radiation damage is mitigated by spreading the radiation dose over many molecules in a crystal. Consequently, most membrane protein structures remain unknown to date. Similarly, determining high-resolution x-ray structures of single, non-periodic biological nanoparticles by x-ray diffraction imaging has been hampered by radiation damage. While cryo-electron microscopy (cryo-EM) has been successful in obtaining high-resolution structural information from large biomolecules and nanoparticles, it requires freezing of the sample as a way to mitigate electron-induced radiation damage and the cryogenic temperatures make it impossible to visualize fast conformational changes. X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow to break this nexus between resolution and absorbed dose by utilizing the ?diffraction-before- destruction? principle and promise imaging at unprecedented spatio-temporal resolution. Since the commissioning of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory only five years ago, both protein structure determination at room temperature to near- atomic resolution by serial-femtosecond nanocrystallography (SFX) and modest resolution structural studies by single nanoparticle diffraction imaging (SPI) have been demonstrated. However, several challenges and limitations remain that need to be overcome to allow more fully utilizing these new light sources for structural biology. The overall objective of this proposed work is to address several of the current technological and methodological challenges in coherent x-ray diffraction imaging of biological samples with XFELs, in particular, in the areas of sample preparation and introduction for membrane proteins and biological nanoparticles. Our work aims to drastically reduce sample consumption and to open up this imaging method to a much broader range of research groups and to samples including a diversity of membrane proteins and other biological nano-objects that are not abundant and/or difficult to crystallize. The proposed work will lay the groundwork for future time-resolved structural studies at XFELs that require efficient use of available sample. If successful, this work would greatly aid our experimental capabilities to study and understand function of protein complexes and biological nanoparticles in a wide range of fields including human health and biosecurity.